The long-term goal of the project is to understand the mechanisms that regulate gene expression during differentiation of the bacterium Bacillus subtilis. Two hypotheses will be tested--that the initiation of differentiation (sporulation) is controlled by the effects of certain metabolites on transcription of a small number of genes and that the sequential appearance of classes of mRNAs during the course of sporulation is due to alterations in the sigma subunit of RNA polymerase. These ideas will be tested by studying transcriptional regulation of cloned genes representative of the different phases of differentiation. After the promoter sites for these genes have been located, they will be mutated by site-specific methods in such a way as to alter regulation. The altered phenotype will be detected by fusion of the mutated promoters to the gene for Escherichia coli Beta-galactosidase. Compensatory mutations will then be sought in unlinked genes whose products may interact with the promoter region. In this way, it may be possible to identify novel regulatory factors and study their mechanisms of action.